Wound healing device and method

ABSTRACT

The invention is directed to a novel wound healing device. In particular, the invention is directed to a novel wound healing device comprising a suture or knitted mesh that has adsorbed onto it novel cellular factor-containing compositions (referred to herein as CFC), including Amnion-derived Cellular Cytokine Solution (referred to herein as ACCS) or Physiologic Cytokine Solutions (herein referred to as PCS), as well as methods of making and uses thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 USC §119(e) of U.S.Provisional Application Nos. 61/461,445, filed Jan. 18, 2011,61/461,951, filed Jan. 25, 2011, and 61/463,185, filed Feb. 14, 2011,the entireties of which are incorporated herein by reference.

FIELD OF THE INVENTION

The field of the invention is directed to a novel wound healing device.In particular, the field of the invention is directed to a novel woundhealing device comprising a suture or a knitted mesh that has adsorbedonto it novel cellular factor-containing compositions (referred toherein as CFC), such CFCs including conditioned medium compositionsobtained from extraembryonic cytokine secreting cells (ECS cells),including Amnion-derived Cellular Cytokine Solution (referred to hereinas ACCS) obtained from Amnion-derived Multipotent Progenitor (AMP)cells, and Physiologic Cytokine Solution (herein referred to as PCS), aswell as methods of making and uses thereof.

BACKGROUND OF THE INVENTION

Protein-based therapeutics are typically more difficult to administer topatients than other pharmaceuticals. Because the efficacy of a proteinis related to its shape, protein-based therapeutics cannot be subjectedto conditions that could cause the unfolding, or denaturing, of theprotein or proteins contained therein. Consequently, special care isnecessary in the preparation, storage, and administration ofprotein-based therapeutics.

In addition to avoiding any denaturation of the protein, it is oftendesirable to be able to control the amount of the protein administeredto a patient over time. This helps to avoid protein concentrationswithin the patient that are undesirably high or low or that fluctuatetoo much from a desired level, and instead helps maintain a steady levelof the therapeutic in the patient. To address this, sustained-release(also known as controlled-release/timed-release, etc.) formulations formany therapeutics, including protein-based therapeutics, have been orare currently in development. Sustained-release protein-basedtherapeutics can be administered by a variety of methods, including butnot limited to oral delivery of tablets or capsules, inhalation ofpowders, implantation, incorporation into a matrix, or topicalapplication of an encapsulated therapeutic from which the protein isgradually released over time.

Surgical sutures are medical devices used to hold body tissues togetherafter an injury or surgery. Most modem sutures are synthetic, includingabsorbable sutures made of, for example, polyglycolic acid, polylacticacid, polydioxanone or caprolactone, all of which are broken down byvarious processes including hydrolysis (polyglycolic acid) andproteolytic enzymatic degradation, and the non-absorbable sutures madeof, for example, nylon, polyester, or polypropylene. Natural materialsused to make sutures include silk and gut. Recently sutures have beencoated with antimicrobial substances to reduce the chances of infection.In addition to being made of different materials, sutures come in veryspecific sizes ranging from #5 (a heavy “braided” suture suitable fororthopedic use) to #11-0 (a fine “monofilament” suture suitable forophthalmic use). Sutures must be strong enough to hold the tissuesecurely but flexible enough to be knotted. They must also behypoallergenic.

There are numerous advantages and disadvantages to both “monofilament”and “braided” sutures. Advantages of the monofilament suture includethat it is very smooth and has a low friction in the tissues, the sutureruns very easily through the tissues, several continuous stitches can bepulled easily through the tissue without causing damage to the tissue,the surface of the suture tends not to harbor infection-causingmicroorganisms so stitch abscesses are less common than with a braidedsuture, and the surface of the suture is less likely to attractsplatelets than a braided suture, which reduces the incidence of thrombusformation. Disadvantages include that monofilament sutures are slipperyfor an assistant to hold, are slippery for knot tying, may kink or snap,require more throws for security than a braided suture, the knots mayslip, which is a disadvantage when relying on a stitch not slipping butis actually an advantage when snugging down 2 throws of a knot, theknots may unravel, are intolerant of the twisting effect of a series ofstitches, tend to form loops more than a braided suture, are morespringy than a braided suture, and have more memory (e.g. resists beingstraightened if it has been coiled up in its packet) than braidedsutures.

The main differences between a monofilament suture and a braided sutureis the higher friction and higher compliance exhibited by the braidedsuture. Advantages of braided sutures include they are less slippery,they are easier to handle, they have less memory (e.g. stretching itwill permanently remove most coils and zigzags), they are easier toknot, the knots tend not to slip, fewer throws are needed on knots, andtheir friction can be used to advantage in subcuticular stitches.Disadvantages include the increased friction means it does not run aseasily as a monofilament suture, even with a single stitch, there is adanger of the pull to overcome friction causing damage to the tissues,pulling the braided suture through more than one stitch at a time ishazardous, except for a subcuticular stitch, there is more chance ofinfection lodging in the braided suture and not being accessible toantibiotics, particularly with a non-absorbable stitch.

In addition to classic sutures, knitted mesh prepared from copolymers ofglycolide and lactide is also suitable for use in the methods of theinvention. The mesh is particularly suitable to reduce or prevent herniaformation following surgery, in particular, abdominal surgery.

Many different suturing techniques exist. The most common technique isthe simple interrupted stitch which is the simplest to perform and iscalled “interrupted” because the suture thread is cut between eachindividual stitch. The vertical and horizontal mattress stitches arealso interrupted but are more complex and specialized for particularsettings. The running or continuous stitch is quicker but risks failingif the suture is cut in just one place; the continuous locking stitch isa more secure version of the running or continuous stitch. The chestdrain stitch and corner stitch are variations of the horizontal mattressstitch. Other stitches include the Figure 8 stitch and subcuticularstitch.

Applicants present herewith for the first time the instant inventionwhose object is to deliver in a controlled manner physiologicallyrelevant growth factors and cytokines at physiologic levels to a woundby adsorbing the compositions onto a suture or a knitted mesh.

BRIEF SUMMARY OF THE INVENTION

It is an object of the instant invention to provide a novel woundhealing device and methods for using the device. Such a wound healingdevice comprises a nonabsorbable or bioerodable braided suture orknitted mesh that has absorbed onto it novel cellular factor-containingcompositions (referred to herein as CFC, including ACCS) or physiologiccytokine solutions (herein referred to as PCS). The CFC, including ACCS,or PCS, contain a complex and unique combination of and physiologiclevels of wound healing cytokines and growth factors found naturally inthe body. Bioerodable braided sutures or knitted mesh are able tobreakdown over time in the body, whereas nonabsorbable sutures persistuntil removed. In accordance with the invention, the bioerodable braidedsutures or knitted mesh are absorbed with the CFC, including ACCS, orPCS, which become trapped in the spaces between the braided filaments ofthe sutures or the knitted mesh. The CFC, including ACCS, or PCS, arethen released into the local wound area over time as a result of acombination of the protein factor release by diffusion and/or theerosion of the bioerodable suture or knitted mesh. Thus, the woundhealing compositions are delivered precisely to the wound area formaximal effect. Because the cellular factors are present in levelscomparable to physiological levels found in the body, they are optimalfor use in therapeutic applications which require intervention tosupport, initiate, replace, accelerate or otherwise influencebiochemical and biological processes involved in the treatment and/orhealing of an injury or wound or, in the case of a knitted mesh, preventor reduce hernia formation. The cellular factors are also releasedslowly over time to provide a continual, consistent physiologic level ofsuch factors to optimize healing and/or recovery. In addition, CFC,including ACCS, or PCS, can be formulated prior to their absorption ontothe suture or knitted mesh. Such formulations may includesustained-release/controlled-release/time-release formulations or theaddition of gelling or thickening agents to improve adsorption onto thesuture or knitted mesh. Details on sustained-release formulations ofCFC, including ACCS, or PCS, can be found in US-2009-0054339-A1,published Feb. 26, 2009, the contents of which is incorporated herein byreference. The CFC, including ACCS, or PCS may also have heparin addedto them prior to soaking the suture, or the sutures may have heparinadsorbed onto them prior to soaking in the CFC, including ACCS, or PCS.Heparin is known to bind to may proteins have heparin binding motifs andmay serve to effect a slow release of the proteins found in the CFC,including ACCS, or PCS. Further, the CFC, including ACCS, or PCS, may belyophilized prior to absorption onto the suture. Such lyophilization mayinclude the addition of other agents, for example collagen. An importantfeature of the novel wound healing device described herein in that it isthe first device disclosed that is capable of delivering numerous woundhealing factors (for example, VEGF, TIMP-1, TIMP-2, PDGF, TGFβ2,Angiogenin) simultaneously, slowly and at physiologic concentrationsdirectly to the wound site.

Accordingly, a first aspect of the invention is a wound healing devicecomprising a cellular factor-containing composition (CFC) adsorbed ontoa braided suture or knitted mesh.

In one embodiment of aspect one the CFC is Amnion-derived CellularCytokine Solution (ACCS).

In another embodiment of aspect one the ACCS is concentrated ACCS.

In still another embodiment of aspect one the CFC is physiologiccytokine solution (PCS).

In yet another embodiment of aspect one the PCS is concentrated PCS.

In another embodiment of aspect one the suture or knitted mesh isnonabsorbable or bioerodable.

In a specific embodiment of aspect one the bioerodable suture or knittedmesh is made of polyglycolic acid, polyglutamic acid, polydioxanone,polylactide or caprolactone, or combinations thereof. In anotherspecific embodiment of aspect one the nonabsorbable suture is made ofnylon, polyester, polypropylene, or silk.

In another specific embodiment of aspect one the CFC comprisesphysiologic concentrations of VEGF, TGFβ2, Angiogenin, PDGF, TIMP-1 andTIMP-2.

In a very specific embodiment of aspect one the physiologicconcentration is ˜5.0-16 ng/mL for VEGF, ˜3.5-4.5 ng/mL for Angiogenin,˜100-165 pg/mL for PDGF, ˜2.5-2.7 ng/mL for TGFβ2, ˜0.68 μmL for TIMP-1and 181.04 μg/mL for TIMP-2.

A second aspect of the invention is a kit comprising the wound healingdevice of aspect one.

A third aspect of the invention is a method of promoting wound healingin a subject in need thereof comprising suturing the wound with thewound healing device of aspect one.

In a particular embodiment of aspect three the suturing is continuoussuturing of the wound.

A fourth aspect of the invention is a method for reducing or preventedhernia formation comprising placing the knitted mesh wound healingdevice of aspect one in a patient during surgery to reduce or preventhernia formation following surgery.

A fifth aspect of the invention is a method for the delivery of amixture of protein factors directly to a wound such that the proteinfactors are delivery simultaneously to the wound, the method comprisingthe step of applying the wound healing device of aspect one to thewound.

Definitions

As defined herein “isolated” refers to material removed from itsoriginal environment and is thus altered “by the hand of man” from itsnatural state.

As used herein, “enriched” means to selectively concentrate or toincrease the amount of one or more materials by elimination of theunwanted materials or selection and separation of desirable materialsfrom a mixture (i.e. separate cells with specific cell markers from aheterogeneous cell population in which not all cells in the populationexpress the marker).

As used herein, the term “substantially purified” means a population ofcells substantially homogeneous for a particular marker or combinationof markers. By substantially homogeneous is meant at least 90%, andpreferably 95% homogeneous for a particular marker or combination ofmarkers.

The term “placenta” as used herein means both preterm and term placenta.

As used herein, the term “totipotent cells” shall have the followingmeaning. In mammals, totipotent cells have the potential to become anycell type in the adult body; any cell type(s) of the extraembryonicmembranes (e.g., placenta). Totipotent cells are the fertilized egg andapproximately the first 4 cells produced by its cleavage.

As used herein, the term “pluripotent stem cells” shall have thefollowing meaning. Pluripotent stem cells are true stem cells with thepotential to make any differentiated cell in the body, but cannotcontribute to making the components of the extraembryonic membraneswhich are derived from the trophoblast. The amnion develops from theepiblast, not the trophoblast. Three types of pluripotent stem cellshave been confirmed to date: Embryonic Stem (ES) Cells (may also betotipotent in primates), Embryonic Germ (EG) Cells, and EmbryonicCarcinoma (EC) Cells. These EC cells can be isolated fromteratocarcinomas, a tumor that occasionally occurs in the gonad of afetus. Unlike the other two, they are usually aneuploid.

As used herein, the term “multipotent stem cells” are true stem cellsbut can only differentiate into a limited number of types. For example,the bone marrow contains multipotent stem cells that give rise to allthe cells of the blood but may not be able to differentiate into othercells types.

As used herein, the term “extraembryonic cytokine-secreting cells” or“ECS cells” means a population of cells derived from the extraembryonictissue which have the characteristic of secreting VEGF, Angiogenin, PDGFand TGFβ2 and the MMP inhibitors TIMP-1 and/or TIMP-2 at physiologicallyrelevant levels in a physiologically relevant temporal manner into theextracellular space or into the surrounding culture media. ECS cellshave not been cultured in the presence of any non-human animalmaterials, making them and cell products derived from them suitable forhuman clinical use as they are not xeno-contaminated. ECS cells may beselected from populations of cells and compositions described in thisapplication and in US2003/0235563, US2004/0161419, US2005/0124003, U.S.Provisional Application Nos. 60/666,949, 60/699,257, 60/742,067,60/813,759, U.S. application Ser. No. 11/333,849, U.S. application Ser.No. 11/392,892, PCTUS06/011392, US2006/0078993, PCT/US00/40052, U.S.Pat. No. 7,045,148, US2004/0048372, and US2003/0032179, the contents ofwhich are incorporated herein by reference in their entirety. ECS cellshave previously been referred to as TSE cells.

As used herein, the term “Amnion-derived Multipotent Progenitor cell” or“AMP cell” means a specific population of cells that are epithelialcells derived from the amnion. AMP cells have the followingcharacteristics. They have not been cultured in the presence of anynon-human animal materials, making them and cell products derived fromthem suitable for human clinical use as they are not xeno-contaminated.AMP cells are cultured in basal medium supplemented with human serumalbumin. In a preferred embodiment, the AMP cells secrete the cytokinesVEGF, Angiogenin, PDGF and TGFβ2 and the MMP inhibitors TIMP-1 and/orTIMP-2. The physiological range of the cytokine or cytokines in theunique combination is as follows: ˜5-16 ng/mL for VEGF, ˜3.5-4.5 ng/mLfor Angiogenin, ˜100-165 pg/mL for PDGF, ˜2.5-2.7 ng/mL for TGFβ2, ˜0.68μg/mL for TIMP-1 and ˜1.04 μg/mL for TIMP-2. The AMP cells mayoptionally express Thymosin β4. AMP cells grow without feeder layers, donot express the protein telomerase and are non-tumorigenic. AMP cells donot express the hematopoietic stem cell marker CD34 protein. The absenceof CD34 positive cells in this population indicates the isolates are notcontaminated with hematopoietic stem cells such as umbilical cord bloodor embryonic fibroblasts. Virtually 100% of the cells react withantibodies to low molecular weight cytokeratins, confirming theirepithelial nature. Freshly isolated amnion-derived cells, from which AMPcells are isolated, will not react with antibodies to thestem/progenitor cell markers c-kit (CD117) and Thy-1 (CD90). Severalprocedures used to obtain cells from full term or pre-term placenta areknown in the art (see, for example, US 2004/0110287; Anker et al., 2005,Stem Cells 22:1338-1345; Ramkumar et al., 1995, Am. J. Ob. Gyn.172:493-500). However, the methods used herein provide improvedcompositions and populations of cells.

By the term “animal-free” when referring to certain compositions, growthconditions, culture media, etc. described herein, is meant that nonon-human animal-derived materials, such as bovine serum, proteins,lipids, carbohydrates, nucleic acids, vitamins, etc., are used in thepreparation, growth, culturing, expansion, storage or formulation of thecertain composition or process. By “no non-human animal-derivedmaterials” is meant that the materials have never been in or in contactwith a non-human animal body or substance so they are notxeno-contaminated. Only clinical grade materials, such as recombinantlyproduced human proteins, are used in the preparation, growth, culturing,expansion, storage and/or formulation of such compositions and/orprocesses.

By the term “expanded”, in reference to cell compositions, means thatthe cell population constitutes a significantly higher concentration ofcells than is obtained using previous methods. For example, the level ofcells per gram of amniotic tissue in expanded compositions of AMP cellsis at least 50 and up to 150 fold higher than the number of amnionepithelial cells in the primary culture after 5 passages, as compared toabout a 20 fold increase in such cells using previous methods. Inanother example, the level of cells per gram of amniotic tissue inexpanded compositions of AMP cells is at least 30 and up to 100 foldhigher than the number of amnion epithelial cells in the primary cultureafter 3 passages. Accordingly, an “expanded” population has at least a 2fold, and up to a 10 fold, improvement in cell numbers per gram ofamniotic tissue over previous methods. The term “expanded” is meant tocover only those situations in which a person has intervened to elevatethe number of the cells.

As used herein, the term “passage” means a cell culture technique inwhich cells growing in culture that have attained confluence or areclose to confluence in a tissue culture vessel are removed from thevessel, diluted with fresh culture media (i.e. diluted 1:5) and placedinto a new tissue culture vessel to allow for their continued growth andviability. For example, cells isolated from the amnion are referred toas primary cells. Such cells are expanded in culture by being grown inthe growth medium described herein. When such primary cells aresubcultured, each round of subculturing is referred to as a passage. Asused herein, “primary culture” means the freshly isolated cellpopulation.

As used herein, “conditioned medium” is a medium in which a specificcell or population of cells has been cultured, and then removed. Whencells are cultured in a medium, they may secrete cellular factors thatcan provide support to or affect the behavior of other cells. Suchfactors include, but are not limited to hormones, cytokines,extracellular matrix (ECM), proteins, vesicles, antibodies, chemokines,receptors, inhibitors and granules. The medium containing the cellularfactors is the conditioned medium. Examples of methods of preparingconditioned media are described in U.S. Pat. No. 6,372,494 which isincorporated by reference in its entirety herein.

As used herein, the term “Amnion-derived Cellular Cytokine Solution” or“ACCS” means conditioned medium that has been derived from AMP cellsthat have been cultured in basal media supplemented with human serumalbumin

The term “physiological level” as used herein means the level that asubstance in a living system is found and that is relevant to the properfunctioning of a biochemical and/or biological process.

As used herein, the term “Physiologic Cytokine Solution” or “PCS”composition means a composition which is not cell-derived and which hasphysiologic concentrations of one or more factors selected from VEGF,Angiogenin, PDGF and TGFβ2 and at least one MMP inhibitor. Examples ofsuitable MMP inhibitors include but are not limited to TIMP-1 andTIMP-2. Details on PCS can be found in U.S. Publication No.US-2009-0054339-A1, the contents of which are incorporated herein byreference.

As used herein, the term “solution” as used in “Amnion-derived CellularCytokine Solution” means a liquid containing dispersed components, i.e.cytokines. The dispersed components may be fully solubilized, partiallysolubilized, suspended or otherwise dispersed in the liquid. Suitableliquids include, but are not limited to, water, osmotic solutions suchas salt and/or sugar solutions, cell culture media, and other aqueous ornon-aqueous solutions.

The term “lysate” as used herein refers to the composition obtained whencells, for example, AMP cells, are lysed and optionally the cellulardebris (e.g., cellular membranes) is removed. This may be achieved bymechanical means, by freezing and thawing, by sonication, by use ofdetergents, such as EDTA, or by enzymatic digestion using, for example,hyaluronidase, dispase, proteases, and nucleases. In some instances, itmay be desirable to lyse the cells and retain the cellular membraneportion and discard the remaining portion of the lysed cells.

As used herein, the term “pooled” means a plurality of compositions thathave been combined to create a new composition having more constant orconsistent characteristics as compared to the non-pooled compositions.For example, pooled ACCS have more constant or consistentcharacteristics compared to non-pooled ACCS. Examples of pooledcompositions include “SP pools” (more than one ACCS collection/oneplacenta), “MP1 pools” (one ACCS collection/placenta, multipleplacentas), and “MP2 pools” (more than one ACCS collection/placenta,multiple placentas).

As used herein, the term “substrate” means a defined coating on asurface that cells attach to, grown on, and/or migrate on. As usedherein, the term “matrix” means a substance that cells grow in or onthat may or may not be defined in its components. The matrix includesboth biological and non-biological substances. As used herein, the term“scaffold” means a three-dimensional (3D) structure (substrate and/ormatrix) that cells grow in or on. It may be composed of biologicalcomponents, synthetic components or a combination of both. Further, itmay be naturally constructed by cells or artificially constructed. Inaddition, the scaffold may contain components that have biologicalactivity under appropriate conditions.

The term “cell product” or “cell products” as used herein refers to anyand all substances made by and secreted from a cell, including but notlimited to, protein factors (i.e. growth factors, differentiationfactors, engraftment factors, cytokines, morphogens, proteases (i.e. topromote endogenous cell delamination, protease inhibitors),extracellular matrix components (i.e. fibronectin, etc.).

The term “therapeutically effective amount” means that amount of atherapeutic agent necessary to achieve a desired physiological effect(i.e. accelerate wound healing).

As used herein, the term “pharmaceutically acceptable” means that thecomponents, in addition to the therapeutic agent, comprising theformulation, are suitable for administration to the patient beingtreated in accordance with the present invention.

As used herein, the term “therapeutic protein” includes a wide range ofbiologically active proteins including, but not limited to, growthfactors, enzymes, hormones, cytokines, inhibitors of cytokines, bloodclotting factors, peptide growth and differentiation factors.

As used herein, the term “tissue” refers to an aggregation of similarlyspecialized cells united in the performance of a particular function.

As used herein, the terms “a” or “an” means one or more; at least one.

As used herein, the term “adjunctive” means jointly, together with, inaddition to, in conjunction with, and the like.

As used herein, the term “co-administer” can include simultaneous orsequential administration of two or more agents.

As used herein, the term “agent” means an active agent or an inactiveagent. By the term “active agent” is meant an agent that is capable ofhaving a physiological effect when administered to a subject.Non-limiting examples of active agents include growth factors,cytokines, antibiotics, cells, conditioned media from cells, etc. By theterm “inactive agent” is meant an agent that does not have aphysiological effect when administered. Such agents may alternatively becalled “pharmaceutically acceptable excipients”. Non-limiting examplesinclude time release capsules and the like.

The terms “parenteral administration” and “administered parenterally”are art-recognized and refer to modes of administration other thanenteral and topical administration, usually by injection, and includes,without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intra-articulare, subcapsular, subarachnoid, intraspinal, epidural,intracerebral and intrasternal injection or infusion.

The term “enteral administration” and “administered enterally” areart-recognized and refer to modes of administration other than enteraland topical administration, usually by oral or rectal routes.

The term “topical administration” and “administered topically” areart-recognized and refer to modes of administration other thanparenteral and enteral administration, usually by application to theskin.

The term “adsorb” as used herein refers to the act of a liquid, gas, ora dissolved substance accumulating on the surface of a solid.

The terms “sustained-release”, “extended-release”, “time-release”,“controlled-release”, or “continuous-release” as used herein means anagent, typically a therapeutic agent or drug, that is released overtime.

The terms “bioerodable” or “bioerosion” as used herein mean acombination of physical (i.e. dissolution) and chemical (i.e. chemicalbond cleavage) processes that result in the breakdown of a substance.

The term “biodegradable” or “biodegradation” as used herein means abiological agent (i.e. an enzyme, microbe or cell) is responsible forthe breakdown of a substance.

The terms “bioresporbable” or “bioabsorptable” as used herein mean theremoval of a breakdown product by cellular activity (i.e. phagocytosis).The term “nonabsorbable” as used herein means that a substance is notbroken down by a chemical process.

“Treatment,” “treat,” or “treating,” as used herein covers any treatmentof a disease or condition of a mammal, particularly a human, andincludes: (a) preventing the disease or condition from occurring in asubject which may be predisposed to the disease or condition but has notyet been diagnosed as having it; (b) inhibiting the disease orcondition, i.e., arresting its development; (c) relieving and orameliorating the disease or condition, i.e., causing regression of thedisease or condition; or (d) curing the disease or condition, i.e.,stopping its development or progression. The population of subjectstreated by the methods of the invention includes subjects suffering fromthe undesirable condition or disease, as well as subjects at risk fordevelopment of the condition or disease.

As used herein, a “wound” is any disruption, from whatever cause, ofnormal anatomy (internal and/or external anatomy) including but notlimited to traumatic injuries such as mechanical (i.e. contusion,penetrating), thermal, chemical, electrical, concussive and incisionalinjuries; elective injuries such as operative surgery and resultantincisional hernias, fistulas, etc.; acute wounds, chronic wounds,infected wounds, and sterile wounds, as well as wounds associated withdisease states (i.e. ulcers caused by diabetic neuropathy or ulcers ofthe gastrointestinal or genitourinary tract). A wound is dynamic and theprocess of healing is a continuum requiring a series of integrated andinterrelated cellular processes that begin at the time of wounding andproceed beyond initial wound closure through arrival at a stable scar.These cellular processes are mediated or modulated by humoral substancesincluding but not limited to cytokines, lymphokines, growth factors, andhormones. In accordance with the subject invention, “wound healing”refers to improving, by some form of intervention, the natural cellularprocesses and humoral substances of tissue repair such that healing isfaster, and/or the resulting healed area has less scaring and/or thewounded area possesses tissue strength that is closer to that ofuninjured tissue and/or the wounded tissue attains some degree offunctional recovery.

As used herein the term “standard animal model for wound healing” refersto any art-accepted animal model for wound healing in which thecompositions of the invention exhibit efficacy as measured byaccelerated wound healing. Non-limiting examples of suitable models aredescribed in Hayward P G, Robson M C: Animal models of woundcontraction. In Barbul A, et al: Clinical and Experimental Approaches toDermal and Epidermal Repair: Normal and Chronic Wounds. John Wiley &Sons, New York, 1990; DelBecarro, et al: The use of specific thromboxaneinhibitors to preserve the dermal microcirculation after burning.Surgery 87: 137-141, 1980; Robson, et al: Increasing dermal perfusionafter burning by decreasing thromboxane production. J Trauma 20:722-725, 1980; Polo, et al: An in vivo model of human proliferativescar. J Surg Res 74: 187-195, 1998.). Skilled artisans are aware ofother suitable models.

DETAILED DESCRIPTION

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either both ofthose included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and” and “the” include plural references unless thecontext clearly dictates otherwise.

Obtaining and Culturing of Cells

ECS Cells—

Various methods for isolating cells from extraembryonic tissue, whichmay then be used to produce the ECS cells of the instant invention aredescribed in the art (see, for example, US2003/0235563, US2004/0161419,US2005/0124003, U.S. Provisional Application Nos. 60/666,949,60/699,257, 60/742,067, 60/813,759, U.S. application Ser. No.11/333,849, U.S. application Ser. No. 11/392,892, PCTUS06/011392,US2006/0078993, PCT/US00/40052, U.S. Pate. No. 7,045,148,US2004/0048372, and US2003/0032179).

Identifying ECS Cells—

Once extraembryonic tissue is isolated, it is necessary to identifywhich cells in the tissue have the characteristics associated with ECScells (see definition above). For example, cells are assayed for theirability to secrete VEGF, Angiogenin, PDGF and TGFβ2 and the MMPinhibitors TIMP-1 and/or TIMP-2 into the extracellular space or intosurrounding culture media. In some instances, it may be difficult orimpossible to detect certain factors using standard assays. This may bebecause certain factors are secreted by the cells at physiologicallevels that are below the level of detection by the assay methods. Itmay also be that the factor(s) is being utilized by the ECS cell and/orby other local cells, thus preventing accumulation at detectable levelsusing standard assays. It is also possible that the temporal manner inwhich the factors are secreted may not coincide with the timing ofsampling.

AMP cell compositions are prepared using the steps of a) recovery of theamnion from the placenta, b) dissociation of the epithelial cells fromthe amniotic membrane using a protease, c) culturing of the cells in abasal medium with the addition of a naturally derived or recombinantlyproduced human protein (i.e. human serum albumin) and no non-humananimal protein; d) selecting AMP cells from the epithelial cell culture,and optionally e) further proliferation of the cells, optionally usingadditional additives and/or growth factors (i.e. recombinant human EGF).Details are contained in US Publication No. 2006-0222634-A1, which isincorporated herein by reference.

Culturing of the AMP Cells—

The cells are cultured in a basal medium. Such medium includes, but isnot limited to, EPILIFE® culture medium for epithelial cells (CascadeBiologicals), OPTI-PRO™ serum-free culture medium, VP-SFM serum-freemedium, IMDM highly enriched basal medium, KNOCKOUT™ DMEM low osmolalitymedium, 293 SFM II defined serum-free medium (all made by Gibco;Invitrogen), HPGM hematopoietic progenitor growth medium, Pro 293S-CDMserum-free medium, Pro 293A-CDM serum-free medium, UltraMDCK™ serum-freemedium (all made by Cambrex), STEMLINE® T-cell expansion medium andSTEMLINE® II hematopoietic stem cell expansion medium (both made bySigma-Aldrich), DMEM culture medium, DMEM/F-12 nutrient mixture growthmedium (both made by Gibco), Ham's F-12 nutrient mixture growth medium,M199 basal culture medium (both made by Sigma-Aldrich), and othercomparable basal media. Such media should either contain human proteinor be supplemented with human protein. As used herein a “human protein”is one that is produced naturally or one that is produced usingrecombinant technology. “Human protein” also is meant to include a humanderivative or preparation thereof, such as human serum, which containshuman protein. In specific embodiments, the basal media is IMDM highlyenriched basal medium, STEMLINE® T-cell expansion medium or STEMLINE® IIhematopoietic stem cell expansion medium, or OPTI-PRO™ serum-freeculture medium, or combinations thereof and the human protein is humanserum albumin is at least 0.5% and up to 10%. In particular embodiments,the human serum albumin is from about 0.5 to about 2%. In a specificembodiment the human albumin is at 0.5%. The human albumin may come froma liquid or a dried (powder) form and includes, but is not limited to,recombinant human serum albumin, PLASBUMIN® normal human serum albuminand PLASMANATE® human blood fraction (both made by TalecrisBiotherapeutics).

In a most preferred embodiment, the cells are cultured using a systemthat is free of non-human animal products to avoid xeno-contamination.In this embodiment, the culture medium is IMDM highly enriched basalmedium , STEMLINE® T-cell expansion medium or STEMLINE® II hematopoieticstem cell expansion medium, OPTI-PRO™ serum-free culture medium, or DMEMculture medium, with human serum albumin (i.e. PLASBUMIN® normal humanserum albumin) added up to amounts of 10%.

The invention further contemplates the use of any of the above basalmedia wherein animal-derived proteins are replaced with recombinanthuman proteins and animal-derived serum, such as BSA, is replaced withhuman serum albumin. In preferred embodiments, the media is serum-freein addition to being animal-free.

Optionally, other factors are used. In one embodiment, epidermal growthfactor (EGF) at a concentration of between 0-1 μg/mL is used. In apreferred embodiment, the EGF concentration is around 10-20 ng/mL. Allsupplements are clinical grade.

Generation of CFC, including ACCS

ECS conditioned medium- is obtained as described below for ACCS, exceptthat ECS cells are used.

Generation of ACC—

The AMP cells of the invention can be used to generate ACCS. In oneembodiment, the AMP cells are isolated as described herein and 1×10⁶cells/mL are seeded into T75 flasks containing between 5-30 mL culturemedium, preferably between 10-25 mL culture medium, and most preferablyabout 10 mL culture medium. The cells are cultured until confluent, themedium is changed and in one embodiment the ACCS is collected 1 daypost-confluence. In another embodiment the medium is changed and ACCS iscollected 2 days post-confluence. In another embodiment the medium ischanged and ACCS is collected 4 days post-confluence. In anotherembodiment the medium is changed and ACCS is collected 5 dayspost-confluence. In a preferred embodiment the medium is changed andACCS is collected 3 days post-confluence. In another preferredembodiment the medium is changed and ACCS is collected 3, 4, 5, 6 ormore days post-confluence. Skilled artisans will recognize that otherembodiments for collecting ACCS from AMP cell cultures, such as usingother tissue culture vessels, including but not limited to cellfactories, flasks, hollow fibers, or suspension culture apparatus, orcollecting ACCS from sub-confluent and/or actively proliferatingcultures, are also contemplated by the methods of the invention. It isalso contemplated by the instant invention that the ACCS becryopreserved following collection. It is also contemplated by theinvention that ACCS be lyophilized following collection. It is alsocontemplated by the invention that ACCS be formulated forsustained-release following collection. Skilled artisans are familiarwith cryopreservation lyophilization, and sustained-release formulationmethodologies.

The ACCS of the invention is characterized by assaying forphysiologically relevant cytokines secreted in the physiologicallyrelevant range of ˜5-16 ng/mL for VEGF, ˜.3.5-4.5 ng/mL for Angiogenin,˜100-165 pg/mL for PDGF, ˜2.5-2.7 ng/mL for TGFβ2, ˜0.68 μg mL forTIMP-1 and ˜1.04 μg/mL for TIMP-2.

It is also contemplated by the invention that ACCS, including pooledACCS, be concentrated prior to use. The appropriate level ofconcentration required will be dependent upon the intended use andtherefore will need to be empirically determined.

Generation of PCS

A non-cellular derived form of CFC termed Physiologic Cytokine Solution(PCS) is generated by combining physiological levels of VEGF,Angiogenin, PDGF, TGFβ2, TIMP-1 and TIMP-2, in a carrier. Thephysiological levels for these cytokines are the same as those found inACCS. Suitable carriers include normal saline, PBS, lactated Ringer'ssolution, cell culture medium, etc. Such compositions are suitable forcryopreservation, lyophilization, sustained-release formulation, and thelike.

It is contemplated that PCS may be produced such that it contains moreconcentrated levels of the factors than those found in CFC, includingACCS, and that it may be subsequently diluted with appropriate diluentprior to use. Appropriate diluents include, without limitation, normalsaline, PBS, lactated Ringer's solution, cell culture media, conditionedcell culture media, water, and the like. Such dilutions may be 1:2, 1:3,1:4, 1:5, 1:10, 1:100, etc. The appropriate concentrations and dilutionsrequired will be dependent upon the intended use and therefore will needto be empirically determined.

The compositions of the invention can be prepared in a variety of waysdepending on the intended use of the compositions. For example, acomposition useful in practicing the invention may be a liquidcomprising an agent of the invention, i.e. CFC, including ACCS, or PCS,in solution, in suspension, or both (solution/suspension). The term“solution/suspension” refers to a liquid composition where a firstportion of the active agent is present in solution and a second portionof the active agent is present in particulate form, in suspension in aliquid matrix. A liquid composition also includes a gel. The liquidcomposition may be aqueous or in the form of an ointment, salve, cream,or the like.

An aqueous suspension or solution/suspension useful for practicing themethods of the invention may contain one or more polymers as suspendingagents. Useful polymers include water-soluble polymers such ascellulosic polymers and water-insoluble polymers such as cross-linkedcarboxyl-containing polymers. An aqueous suspension orsolution/suspension of the present invention is preferably viscous ormuco-adhesive, or even more preferably, both viscous and muco-adhesive.

Alternative Formulation of CFC, including ACCS, or PCS

The CFC, including ACCS, or PCS, may be formulated assustained-release/controlled-release/timed-release compositions. Skilledartisans are familiar with methodologies to create such compositions oftherapeutic agents, including protein-based therapeutic agents such asCFC, including ACCS, or PCS.

Sustained-release/controlled-release/timed-release CFC, including ACCS,or PCS may be made by any of the methods described herein. For example,multivesicular liposome formulation technology is useful for thesustained-release of protein and peptide therapeutics. Qui, J., et al,(ACTA Pharmacol Sin, 2005, 26(11):1395-401) describe this methodologyfor the formulation of sustained-release interferon alpha-2b. Vyas, S.P., et al, (Drug Dev Ind Pharm, 2006, 32(6):699-707) describeencapsulating pegylated interferon alpha in multivesicular liposomes.CFC, including ACCS, or PCS are suitable for use in multivesicularliposome sustained-release formulation.

Nanoparticle technology is also useful for creating CFC, including ACCS,or PCS sustained-release/controlled-release/timed-release compositions.For example, Packhaeuser, C. B., et al, (J Control Release, 2007,123(2):131-40) describe biodegradable parenteral depot systems based oninsulin loaded dialkylaminoalkyl-amine-poly(vinylalcohol)-g-poly(lactide-co-glycolide) nanoparticules and conclude thatnanoparticle-based depots are suitable candidates for the design ofcontrolled-release devices for bioactive macromolecules (i.e. proteins).Dailey, L. A., et al, (Pharm Res 2003, 20(12):2011-20) describesurfactant-free, biodegradable nanoparticles for aerosol therapy whichis based on the branched polymers DEAPA-PVAL-g-PLGA and conclude thatDEAPA-PVAL-g-PLGA are versatile drug delivery systems. CFC, includingACCS, or PCS are suitable for use in nanoparticle-basedsustained-release formulations.

Polymer-based sustained-release formulations are also very useful. Chan,Y. P., et al, (Expert Opin Drug Deliv, 2007, 4(4):441-51) provide areview of the Medusa system (Flamel Technologies), which is used forsustained-release of protein and peptide therapies. Thus far, the Medusasystem has been applied to subcutaneous injection of IL-2 andIFN-alpha(2b), in animal models (rats, dogs, monkeys), and in clinicaltrials in renal cancer (IL-2) and hepatitis C (IFN-alpha(2b)) patients.Chavanpatil, M. D., et al, (Pharm Res, 2007, 24(4):803-10) describesurfactant-polymer nanoparticles as a novel platform for sustained andenhanced cellular delivery of water-soluble molecules. Takeuchi, H., etal, (Adv Drug Deliv Res, 2001, 47(1):39-54) describe mucoadhesivenanoparticulate systems for peptide drug delivery, including liposomesand polymeric nanoparticles. Wong, H. L., et al, (Pharm Res, 2006,23(7):1574-85) describe a new polymer-lipid hybrid system which has beenshown to increase cytotoxicity of doxorubicin againstmultidrug-resistant breast cancer cells. CFC, including ACCS, or PCS aresuitable for use in the aforementioned sustained-release formulationmethodologies.

In addition, other sustained-release methodologies familiar to skilledartisans, while not specifically described herein, are also suitable foruse with the CFC, including ACCS, or PCS compositions.

Pharmaceutical Compositions—

The present invention provides pharmaceutical compositions of CFC,including ACCS, or PCS and a pharmaceutically acceptable carrier. Theterm “pharmaceutically acceptable” means approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly, in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the composition isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. Examples of suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin, and still othersare familiar to skilled artisans.

The pharmaceutical compositions of the invention can be formulated asneutral or salt forms. Pharmaceutically acceptable salts include thoseformed with free amino groups such as those derived from hydrochloric,phosphoric, acetic, oxalic, tartaric acids, etc., and those formed withfree carboxyl groups such as those derived from sodium, potassium,ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine,2-ethylamino ethanol, histidine, procaine, etc.

Treatment Kits—

The invention also provides for an article of manufacture comprisingpackaging material and a pharmaceutical composition of the inventioncontained within the packaging material, wherein the pharmaceuticalcomposition comprises compositions of CFC, including ACCS, or PCS, andnonabsorbable or bioerodable braided suture or knitted mesh (the WoundHealing Device). The packaging material comprises a label or packageinsert which indicates that the Wound Healing Device can be used forpromoting wound healing and/or reducing or preventing hernia formationin a subject in need thereof.

One of skill in the art may readily determine the appropriateconcentration, or dose, of the CFC, including ACCS, or PCS, for aparticular purpose. The skilled artisan will recognize that a preferreddose is one which produces a therapeutic effect, such as promoting woundhealing and/or reducing or preventing hernia formation, in a patient inneed thereof. Of course, proper doses of the CFC, including ACCS, orPCS, will require empirical determination at time of use based onseveral variables including but not limited to the severity and type ofinjury or wound being treated; patient age, weight, sex, health; othermedications and treatments being administered to the patient; and thelike. One of skill in the art will also recognize that number of doses(dosing regimen) to be administered needs also to be empiricallydetermined based on, for example, severity and type of disease, injury,disorder or condition being treated. In a preferred embodiment, one doseis sufficient.

The present invention provides a method of promoting wound healingand/or reducing or preventing hernia formation by administering atherapeutically effective dose of CFC, including ACCS, or PCS, to asubject via the Wound Healing Device. By “therapeutically effectiveamount” is meant the dose of CFC, including ACCS, or PCS, which issufficient to elicit a therapeutic effect. Thus, the concentration ofCFC, including ACCS, or PCS, in an administered dose unit in accordancewith the present invention is effective in, for example, promoting woundhealing and/or reducing or preventing hernia formation.

In further embodiments of the present invention, it may be desirable toco-administer other agents, including active agents and/or inactiveagents, with the Wound Healing Device to promote wound healing and/orreducing or preventing hernia formation. Active agents include but arenot limited to cytokines, chemokines, antibodies, inhibitors,antibiotics, anti-fungals, anti-virals, immunosuppressive agents, othercell types, and the like. Inactive agents include carriers, diluents,stabilizers, gelling agents, thickening agents (i.e. human serumalbumin, hyaluronic acid), delivery vehicles, ECMs (natural andsynthetic), scaffolds, collagen, and the like. When the Wound HealingDevice is administered conjointly with other pharmaceutically activeagents, even less of the CFC, including ACCS, or PCS, in the WoundHealing Device may be needed to be therapeutically effective.

The timing of administration of the Wound Healing Device will dependupon the type and severity of the injury or wound being treated. In apreferred embodiment, the Wound Healing Device is administered as soonas possible after the injury or wound occurs.

Skilled artisans will recognize that any and all of the standard methodsand modalities for treating injuries and wounds currently in clinicalpractice and clinical development are suitable for practicing themethods of the invention. Routes of administration, formulation,co-administration with other agents (if appropriate) and the like arediscussed in detail elsewhere herein.

Exemplary Therapeutic Uses of the Wound Healing Device

Wound Healing—

The Wound Healing Device of the present invention is effective inaccelerating wound healing of wounds caused by a number of sources,including but not limited to incisional, compression, thermal,penetrating, concussive, acute, chronic, infected, and sterile injuries.The instant invention is based upon the discovery that ACCS, which isadsorbed onto the suture or knitted mesh, can accelerate the woundhealing process for all wound types. Accordingly, using the WoundHealing Device, all wound types, mechanical or thermal, acute orchronic, infected or sterile, may undergo healing more rapidly thansimilar wounds left to heal naturally or which are treated withcurrently available methods. In addition, the Wound Healing Devicecomprising a knitted mesh is suitable for use to prevent herniaformation following surgery, particularly abdominal surgery. A“therapeutically effective amount” of a therapeutic agent within themeaning of the present invention will be determined by a patient'sattending physician or veterinarian. Such amounts are readilyascertained by one of ordinary skill in the art and will enableaccelerated wound healing when administered in accordance with thepresent invention. Factors which influence what a therapeuticallyeffective amount will be include, the specific activity of thetherapeutic agent being used, the wound type (mechanical or thermal,full or partial thickness, surgical, etc.), the size of the wound, thewound's depth (if full thickness), the absence or presence of infection,time elapsed since the injury's infliction, and the age, physicalcondition, existence of other disease states (i.e. obesity and/ordiabetes), and nutritional status of the patient. Additionally, othermedication the patient may be receiving will effect the determination ofthe therapeutically effective amount of the therapeutic agent toadminister.

Examples

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the compositions and methods of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees centigrade, and pressure isat or near atmospheric.

Example 1 Preparation of AMP Cell Compositions

Amnion epithelial cells were dissociated from starting amniotic membraneusing the dissociation agents PXXIII. The average weight range of anamnion was 18-27 g. The number of cells recovered per g of amnion wasabout 10-15×10⁶ for dissociation with PXXIII.

Method of obtaining selected AMP cells—Amnion epithelial cells wereplated immediately upon isolation from the amnion. After ˜2 days inculture non-adherent cells were removed and the adherent cells werekept. This attachment to a plastic tissue culture vessel is theselection method used to obtain the desired population of AMP cells.Adherent and non-adherent AMP cells appear to have a similar cellsurface marker expression profile but the adherent cells have greaterviability and are the desired population of cells. Adherent AMP cellswere cultured in basal medium supplemented with human serum albuminuntil they reached ˜120,000-150,000 cells/cm². At this point, thecultures were confluent. Suitable cell cultures will reach this numberof cells between ˜5-14 days. Attaining this criterion is an indicator ofthe proliferative potential of the AMP cells and cells that do notachieve this criterion are not selected for further analysis and use.Once the AMP cells reached ˜120,000-150,000 cells/cm², they werecollected and cryopreserved. This collection time point is called p0.

Example 2 Generation of ACCS

The AMP cells of the invention can be used to generate ACCS, includingpooled ACCS. The AMP cells were isolated as described above and ˜1×10⁶cells/mL were seeded into T75 flasks containing ˜10 mL culture medium asdescribed above. The cells were cultured until confluent, the medium waschanged and ACCS was collected 3 days post-confluence. Optionally, theACCS is collected again after 3 days, and optionally again after 3 days.Skilled artisans will recognize that other embodiments for collectingACCS from confluent cultures, such as using other tissue culturevessels, including but not limited to cell factories, flasks, hollowfibers, or suspension culture apparatus, etc. are also contemplated bythe methods of the invention (see Detailed Description above). It isalso contemplated by the instant invention that the ACCS becryopreserved, lyophilized, irradiated or formulated forsustained-release following collection. It is also contemplated thatACCS be collected at different time points (see Detailed Description fordetails).

Example 3 Generation of Pooled ACCS

ACCS was obtained essentially as described above. In certainembodiments, ACCS was collected multiple times from an AMP culturederived from one placenta and these multiple ACCS collections werepooled together. Such pools are referred to as “SP pools” (more than oneACCS collection/one placenta). In another embodiment, AMP cultures werederived from several placentas, i.e. from 5 or 10 placentas. The AMPcells from each placenta were cultured and one ACCS collection from eachculture was collected and then they were all pooled. These pools aretermed “MP1 pools” (one ACCS collection/placenta, multiple placentas).In yet another embodiment, AMP cell cultures were derived from severalplacentas, i.e. from 5 or 10 placentas. The AMP cells from each placentawere cultured and more than one ACCS collection was performed from eachAMP cell culture and then pooled. These pools are termed “MP2 pools”(more than one ACCS collection/placenta, multiple placentas).

Example 4 Production of PCS

The following PCS compositions are produced:

Composition A: VEGF and TIMP-1; Composition B: VEGF, Angiogenin andTIMP-1; Composition C: VEGF, Angiogenin, PDGF-BB and TIMP-1; CompositionD: VEGF, Angiogenin, PDGF-BB, TGFβ2 and TIMP-1; Composition E: VEGF andTIMP-2; Composition F: VEGF, Angiogenin and TIMP-2; Composition G: VEGF,Angiogenin, PDGF-BB and TIMP-2; Composition H: VEGF, Angiogenin,PDGF-BB, TGFβ2 and TIMP-2; Composition I: VEGF, TIMP-1 and TIMP-2;Composition J: VEGF, Angiogenin, TIMP-1 and TIMP-2; Composition K: VEGF,Angiogenin, PDGF-BB, TIMP-1 and TIMP-2; Composition L: VEGF, Angiogenin,PDGF-BB, TGFβ2, TIMP-1 and TIMP-2; Composition M: Angiogenin and TIMP-1;Composition N: Angiogenin, PDGF-BB and TIMP-1; Composition 0:Angiogenin, PDGF-BB, TGFβ2 and TIMP-1; Composition P: Angiogenin andTIMP-2; Composition Q: Angiogenin, PDGF-BB and TIMP-2; Composition R:Angiogenin, PDGF-BB, TGFβ2 and TIMP-2; Composition S: Angiogenin,PDGF-BB, TGFβ2, TIMP-1 and TIMP-2; Composition T: PDGF-BB and TIMP-1;Composition U: PDGF-BB, TGFβ2 and TIMP-1; Composition V: PDGF-BB andTIMP-2; Composition W: PDGF-BB, TGFβ2 and TIMP-2; Composition X:PDGF-BB, TIMP-1 and TIMP-2; Composition Y: PDGF-BB, TGFβ2, TIMP-1 andTIMP-2.

VEGF, Angiogenin, PDGF-BB, TGFβ2, TIMP-1 and TIMP-2 are added at thefollowing physiologic levels: ˜5-16 ng/mL for VEGF, ˜3.5-4.5 ng/mL forAngiogenin, ˜100-165 pg/mL for PDGF, ˜2.5-2.7 ng/mL for TGFβ2, ˜0.68 μgmL for TIMP-1 and ˜1.04 μg/mL for TIMP-2.

VEGF may be obtained from Invitrogen, catalog #PHG0144, PHG0145,PHG0146, PHG0141 or PHG0143; Angiogenin may be obtained from R&DSystems, catalog #265-AN-050 or 265-AN-250; PDGF-BB may be obtained fromInvitrogen, catalog #PHG0044, #PHG0045, #PHG0046, #PHG0041, #PHG0043;TGFβ2 may be obtained from Invitrogen, catalog #PHG9114; TIMP-1 may beobtained from R&D Systems, catalog #970-TM-010; and TIMP-2 may beobtained from R&D Systems, catalog #971-TM-010.

VEGF, Angiogenin, PDGF-BB, TGFβ2, TIMP-1 and TIMP-2 are added to acarrier such as normal saline, PBS, lactated Ringer's solution, cellculture media, or other suitable aqueous solutions known to skilledartisans.

Example 5 Production of Wound Healing Device

The Wound Healing Device of the invention is made by adsorbing onto anonabsorbable or bioerodable braided suture or knitted mesh CFC,including ACCS, concentrated ACCS, PCS, or concentrated PCS. Suitablebraided sutures are available commercially (non-limiting examplesinclude bioerodable sutures such as DEXON™ S braided absorbable sutureand POLYSORB™ coated braided suture, and nonabsorbable sutures such asSURGILON™, SURGIDAC™, TI•CRON™, Covidien, Norwalk, Conn. 06856, USA).Suitable knitted mesh is available commercially from Ethicon, Inc.,Somerville, N.J. (for example, Vicryl™ Knitted Mesh (Polyglycan 910).

The CFC, including ACCS, concentrated ACCS, PCS, or concentrated PCS,may be adsorbed as a liquid onto the braided sutures or knitted mesh ormay be subjected to alternative formulation prior to absorption.Alterative formulations includesustained-release/controlled-release/timed-release as described indetail elsewhere in the specification. Also contemplated areformulations in which gelling or thickening agents such as human serumalbumin, hyaluronic acid, or collagen are added to the CFC, includingACCS, concentrated ACCS, PCS, or concentrated PCS. Such agents increasethe ability of the CFC, including ACCS, concentrated ACCS, PCS, orconcentrated PCS to adsorbed onto the surface or be released from thesurface.

The Wound Healing Device may be packaged with the suture or knitted meshand the CFC, including ACCS, concentrated ACCS, PCS, or concentrated PCSalready adsorbed onto it or it may be packaged with the two componentspackaged separately and then combined just prior to use.

Example 7 Adsorption of ACCS onto Sutures and Subsequent Release ofProteins Off of the Sutures

The purpose of this experiment was to evaluate the ability of varioussutures to adsorb ACCS and to evaluate the ability for proteinscontained in ACCS to elute from the sutures. Two types of sutures weretested in this study: Size 1 DEXON™ S (Covidien, Norwalk, Conn.) andSize 1 MERSILENE™ (Ethicon, Inc., Somerville, N.J.). Both sutures wereuncoated since it was determined in a previous experiment that coatedsutures inhibit the adsorption of liquid. DEXON™ is an absorbable sutureand MERSILENE™ is a non-absorbable polyester suture. Each type of suturewas placed in 50× concentrated ACCS for 30 minutes. The sutures werethen rinsed for various time periods and the rinses were analyzed byELISA to determine the amount of ACCS adsorbed or released. ELISAresults for Angiogenin and TIMP-2 showed that both types of sutureadsorb and release the ACCS proteins. DEXON™ S appeared to release moreACCS into the rinses. The exact amount of adsorption was unclear in thisexperiment, however, it appears that each 10 cm piece of suture releasedbetween 5-6.5 μL of 50× ACCS in 15 minutes.

Example 6 Evaluation of the Wound Healing Device in an Animal Model

One object of the invention is to decrease wound failure in surgical andtraumatic injuries by treating these acute wounds with the wound healingdevice. Mechanically, failing abdominal laparotomy incisions formincisional hernias. Clinically, this manifests as defects in themusculo-tendinous-peritoneal layer of the abdominal wall. Thus focusingon muscle, fascial and skin healing is important. The serious clinicalconsequences of such failures are acute abdominal wall dehiscence andevisceration, the incarceration and obstruction of peritoneal viscera,loss of the ability of the abdominal wall to maintain torso posture, andchronic pain.

The purpose of the experiment is to evaluate the ability of the woundhealing device to reduce or prevent hernia formation in an animal modelof incisional hernia. The experiment will utilize male Sprague-Dawleyrats which will be subjected to abdominal incision and then treated withthe wound healing device or appropriate controls. At the end of thestudy period, post euthanasia, a 5×10 cm section of the abdominal wallwill be excised from each animal. The muscle will be stretched out andpinned on a dissecting board (on each of the four corners) with theperitoneal side facing upwards. A ruler, along with individualidentifiers, will be placed next to each sample and a standardizedpicture will be taken equi-distance from each sample to ensureappropriate scaling. The presence or absence of hernia will be observedand hernia size will be measured

The present invention may be embodied in other specific forms withoutdeparting from the spirit or essential attributes thereof. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

Throughout the specification various publications have been referred to.It is intended that each publication be incorporated by reference in itsentirety into this specification.

1. A wound healing device comprising a cellular factor-containingcomposition (CFC) adsorbed onto a suture or knitted mesh.
 2. The woundhealing device of claim 1 wherein the CFC is Amnion-derived CellularCytokine Solution (ACCS).
 3. The wound healing device of claim 2 whereinthe ACCS is concentrated ACCS.
 4. The wound healing device of claim 1wherein the CFC is physiologic cytokine solution (PCS).
 5. The woundhealing device of claim 4 wherein the PCS is concentrated PCS.
 6. Thewound healing device of claim 1 wherein the suture or knitted mesh isbraided.
 7. The wound healing device of claim 1 wherein the suture isnonabsorbable or bioerodable and the knitted mesh is bioerodable.
 8. Thewound healing device of claim 7 wherein the bioerodable suture orknitted mesh is made of polyglycolic acid, polyglutamic acid,polydioxanone, polylactide, or caprolactone.
 9. The wound healing deviceof claim 7 wherein the nonabsorbable suture is made of nylon, polyester,polypropylene or silk.
 10. The wound healing device of claim 1 whereinthe CFC comprises physiologic concentrations of VEGF, TGFβ2, Angiogenin,PDGF, TIMP-1 and TIMP-2.
 11. The wound healing device of claim 10wherein the physiologic concentration is ˜5.0-16 ng/mL for VEGF,˜3.5-4.5 ng/mL for Angiogenin, ˜100-165 pg/mL for PDGF, ˜2.5-2.7 ng/mLfor TGFβ2, ˜0.68 μg mL for TIMP-1 and ˜1.04 μg/mL for TIMP-2.
 12. A kitcomprising the wound healing device of claim
 11. 13. A method ofpromoting wound healing in a subject in need thereof comprising suturingthe wound with the wound healing device of claim
 1. 14. The method ofclaim 13 wherein the suturing is continuous suturing of the wound.
 15. Amethod of reducing or preventing hernia formation in a subject in needthereof comprising placing a wound healing device comprising a cellularfactor-containing composition (CFC) adsorbed onto a knitted mesh on thewound following surgery.
 16. A method for the delivery of a mixture ofprotein factors directly to a wound such that the protein factors aredelivery simultaneously to the wound, the method comprising the step ofapplying the wound healing device of claim 1 to the wound.